The ability to rapidly and sensitively identify bacterial pathogens in clinical samples is essential to timely and cost-effective initiation of appropriate therapy. Given their inherent ability to overcome assay time and sensitivity limitations, molecular methods can significantly affect the efficacy of in vitro diagnostics. To date however, no single molecular approach has replaced or supplemented the majority of traditional culture tests in clinical microbiology laboratories. We propose the development of an integrated in vitro molecular diagnostic assay through the synergistic combination of existing complementary techniques to challenge the current nucleic acid-based diagnostic paradigm. Our approach uses three tools: advanced peptide nucleic acid-based (PNA) technology for highly specific and selective dsDNA sequence targeting, sequence amplification using a rolling circle mechanism (RCA) ensuring high sensitivity, and a label-free microarray-amenable detection technique for the rapid imaging of DNA nanoparticles amplified directly on a glass surface for screening applications. Peptide nucleic acid technology affords the creation of unique, pathogen-specific PNA-DNA constructs in dsDNA, which allows selective targeting of genomic DNA sites under isothermal non-denaturing conditions. The hybridization of specially designed nucleic acid primers to this construct allows for selective RCA amplification of the target sequence and minimizing of amplification of non-specific genomic material. By augmenting RCA to occur directly on-surface we propose sensing of the amplified pathogen-specific probes through the use of a nanoparticle imaging system, capable of detecting amplified products across a large oligomeric array. The single particle imaging capability of this technique allows very high sensitivity with substantial multiplexing for screening numerous interactions simultaneously. We believe that the integration of these technologies can improve the sensitivity and specificity of a pathogen screening assay over currently available molecular approaches because of redundant and selective steps in sequence targeting and amplification. Moreover, because all steps of the procedure can be performed under isothermal conditions the instrumentation necessary for thermocycling can be simplified or eliminated. We propose a strategy that is expected to overcome a long-standing challenge: the sensitive detection of pathogenic microorganisms without time-consuming culture amplification. The developed technology will yield an unconventional, non-PCR assay for expedient identification of bacterial and viral infections not achievable by alternative techniques. The requested funding would drive this integrated technology toward clinically relevant application and advance the transition from a sensitive research tool to a medical diagnostic technique.